We are investigating how heparan sulfate influences FGFR signaling in specific progenitor cell types in the epithelium. The exquisite control of growth factor function by HS is dictated by the tremendous structural heterogeneity of its sulfated modifications. It is not known how specific HS structures control growth factor-dependent progenitor expansion during organogenesis. We used bioengineered 3-O-sulfated-HS to investigate HS function. Defining the saccharide sequences that determine the selectivity and specificity of their function will facilitate the synthesis of HS mimetics to specifically increase progenitor expansion in vitro. Understanding how specific HS structures regulate FGFR signaling will be useful to target or direct FGFR signaling to regenerate tissue. We also continue an ongoing project that involves generating knockout mice of the Hs3st isoforms to create a genetic toolkit to understand the basic biology of how 3-O-sulfation influences organ development and homeostasis. HS-3-O-sulfotransferase enzymes generate two types of 3-O-sulfated HS epitopes that bind antithrombin and a herpes simplex virus coat protein. We previously showed that 3-O-sulfated HS controls epithelial progenitor cell proliferation and expansion during SMG development. The murine submandibular gland (SMG) expresses four 3-O-sulfotransferase enzymes (Hs3st1, Hs3st3a1, Hs3st3b1, Hs3st6). Our goal is to use genetics to understand the 3-O-sulfated code of HS. This information will be useful to manipulate cellular specificity of HS-binding growth factors and fine-tune biological responses, enhancing progenitor expansion for tissue regeneration. While we focus on the salivary gland, the mouse phenotypes direct us to investigate earlier stages of embryo development and to compare other organs that are affected. These mice are important tools to analyze 3-O-sulfation in salivary gland progenitors and to better understand the fine tuning of cellular responses to FGFRs and HS modifications.